Repeat sequence chromosome specific nucleic acid probes and methods of preparing and using

Abstract

A primer directed DNA amplification method to isolate efficiently chromosome-specific repeated DNA wherein degenerate oligonucleotide primers are used is disclosed. The probes produced are a heterogeneous mixture that can be used with blocking DNA as a chromosome-specific staining reagent, and/or the elements of the mixture can be screened for high specificity, size and/or high degree of repetition among other parameters. The degenerate primers are sets of primers that vary in sequence but are substantially complementary to highly repeated nucleic acid sequences, preferably clustered within the template DNA, for example, pericentromeric alpha satellite repeat sequences. The template DNA is preferably chromosome-specific. Exemplary primers ard probes are disclosed. The probes of this invention can be used to determine the number of chromosomes of a specific type in metaphase spreads, in germ line and/or somatic cell interphase nuclei, micronuclei and/or in tissue sections. Also provided is a method to select arbitrarily repeat sequence probes that can be screened for chromosome-specificity.

We claim: 
     
       1. A method of preparing chromosome-specific repeat sequence nucleic acid probes comprising: hybridizing a first set of degenerate oligonucleotide primers to DNA strands of repeat sequence units in a template DNA that is chromosome-specific;   hybridizing a second set of degenerate oligonucleotide primers to the DNA strands of said repeat sequence units that are complementary to the DNA strands to which the first set of primers hybridize, such that, if said complementary strands of said repeat sequence units were aligned, and if a primer from both the first and second sets hybridizes within a repeat sequence unit, the 5' end of the primer from the first set faces on the complementary strand the 5' end of the primer from the second set; and such that, if said complementary strands were aligned, the hybridization sites of primers from said first and second sets on their respective complementary strands are within a distance of between about 20 base pairs (bp) to about 5 kilobases (kb);   amplifying the template DNA by a polymerase chain reaction (PCR) method; and   producing chromosome-specific repeat sequence nucleic acid probes by performing one or more of the following steps as necessary: (a) selecting from the PCR products dimers or higher order repeats; (b) adding to said PCR products unlabeled blocking DNA, wherein said blocking DNA is human genomic DNA, Cot 1 DNA and/or human alphoid DNA; and (c) cloning said PCR products and screening the clones for chromosome-specificity, repeat content and/or size;   wherein said first and second primer sets are selected from the group consisting of: Jun1 (SEQ ID NO: 15), WA1 (SEQ ID NO: 1), WA2 (SEQ ID NO: 2), WA11 (SEQ ID NO: 3), and WA12 (SEQ ID NO: 4).   
     
     
       2. A method according to claim 1 wherein said first and second primer sets are selected from the group consisting of WA1 (SEQ ID NO: 1), WA2 (SEQ ID NO: 2), WA11 (SEQ ID NO: 3) and WA12 (SEQ ID NO: 4). 
     
     
       3. A method according to claim 1 wherein said first set of degenerate oligonucleotide primers is the same as said second set of oligonucleotide primers. 
     
     
       4. A method according to claim 3 wherein said first and second primer sets are Jun1 (SEQ ID NO: 15). 
     
     
       5. A chromosome-specific repeat sequence probe prepared by the method of claim 1 and labeled during the amplifying step or thereafter. 
     
     
       6. A method of preparing and amplifying chromosome-specific repeat sequence nucleic acid probes comprising: hybridizing a first set of degenerate oligonucleotide primers to DNA strands of repeat sequence units in a template DNA that is chromosome-specific;   hybridizing a second set of degenerate oligonucleotide primers to the DNA strands of said repeat sequence units that are complementary to the DNA strands to which the first set of primers hybridize, such that, if said complementary strands of said repeat sequence units were aligned, and if a primer from both the first and second sets hybridizes within a repeat sequence unit, the 5' end of the primer from the first set faces on the complementary strand the 5' end of the primer from the second set; and such that, if said complementary strands were aligned, the hybridization sites of primers from said first and second sets on their respective complementary strands are within a distance of between about 20 base pairs (bp) to about 5 kilobases (kb);   amplifying the template DNA by a polymerase chain reaction (PCR) method;   inserting the products of said PCR amplification into cloning vectors; cloning said PCR products; and screening the clones for chromosome-specificity, repeat content and/or size to select chromosome-specific repeat sequence probes; selecting clones from the group consisting of pBS609-51 which contains SEQ ID NO: 17 as an insert, and pBS609-52 which contains SEQ ID NO: 18 as an insert; and   amplifying the selected clones using PCR.   
     
     
       7. A method according to claim 6 wherein the primers used to amplify said selected clones are nondegenerate. 
     
     
       8. A chromosome-specific repeat sequence probe prepared by the method of claim 6 and labeled during the step of amplifying the selected clones or thereafter. 
     
     
       9. A method of preparing chromosome-specific repeat sequence DNA probes by arbitrary selection of repeat DNA sequences from human genomic DNA comprising the steps of: annealing to human genomic template DNA containing CAGG repeat sequences the degenerate primer Jun1 (SEQ ID NO: 15);   amplifying said template DNA by a polymerase chain reaction (PCR); and   producing chromosome-specific repeat sequence nucleic acid probes by performing one or more of the following steps: (a) selecting from the PCR products dimers or higher order repeats; (b) adding to said PCR products unlabeled blocking DNA, wherein said blocking DNA is human genomic DNA, Cot 1 DNA and/or human alphoid DNA; and (c) cloning said PCR products and screening the clones for chromosome-specificity, repeat content and/or size.   
     
     
       10. A method according to claim 9 wherein said template DNA is chromosome-specific. 
     
     
       11. A chromosome-specific probe prepared by the method of claim 9 and labeled during the amplifying step or thereafter. 
     
     
       12. A composition of matter consisting of an alpha repeat sequence probe specific for chromosome-10 centromeres having the nucleotide sequence for the insert of pBS609-51 (SEQ ID NO: 17) or pBS609-52 (SEQ ID NO: 18) as shown in Table II. 
     
     
       13. A composition of matter comprising a degenerate oligonucleotide set of primers selected from the group consisting of: WA1 (SEQ ID NO: 1), WA2 (SEQ ID NO: 2), WA11 (SEQ ID NO: 3), WA12 (SEQ ID NO: 4), and Jun1 (SEQ ID NO: 15).